Isolation and Characterization of Chi-like Salmonella Bacteriophages Infecting Two Salmonella enterica Serovars, Typhimurium and Enteritidis

Salmonella enterica Serovar Typhimurium and Salmonella enterica Serovar Enteritidis are well-known pathogens that cause foodborne diseases in humans. The emergence of antibiotic-resistant Salmonella serovars has caused serious public health problems worldwide. In this study, two lysogenic phages, STP11 and SEP13, were isolated from a wastewater treatment plant in Jeddah, KSA. Transmission electron microscopic images revealed that both phages are new members of the genus “Chivirus” within the family Siphoviridae. Both STP11 and SEP13 had a lysis time of 90 min with burst sizes of 176 and 170 PFU/cell, respectively. The two phages were thermostable (0 °C ≤ temperature < 70 °C) and pH tolerant at 3 ≤ pH < 11. STP11 showed lytic activity for approximately 42.8% (n = 6), while SEP13 showed against 35.7% (n = 5) of the tested bacterial strains. STP11 and STP13 have linear dsDNA genomes consisting of 58,890 bp and 58,893 bp nucleotide sequences with G + C contents of 57% and 56.5%, respectively. Bioinformatics analysis revealed that the genomes of phages STP11 and SEP13 contained 70 and 71 ORFs, respectively. No gene encoding tRNA was detected in their genome. Of the 70 putative ORFs of phage STP11, 27 (38.6%) were assigned to functional genes and 43 (61.4%) were annotated as hypothetical proteins. Similarly, 29 (40.8%) of the 71 putative ORFs of phage SEP13 were annotated as functional genes, whereas the remaining 42 (59.2%) were assigned as nonfunctional proteins. Phylogenetic analysis of the whole genome sequence demonstrated that the isolated phages are closely related to Chi-like Salmonella viruses.


Antibiotic Sensitivity Testing
The sensitivity of the host bacterium and its antibiotic resistance profile was assessed by the disk diffusion method, as described previously [38], following the CLSI guidelines [39]. The antibiotics used in this test were purchased from Oxoid ™ Ltd.

Enrichment and Isolation of Phages
The raw wastewater samples were gathered from the wastewater treatment plant in Jeddah city, Kingdom of Saudi Arabia, as elucidated formerly, with slight modifications [40]. In brief, a 20 mL wastewater sample was centrifuged at 8000× g for 12 min at 25 • C and filtered through a 0.22-µm syringe filter (Fischer Scientific, Ottawa, ON, Canada) to remove bacteria, large particulates, and debris.
Bacteriophage enrichment, isolation, and purification were achieved as described previously [41,42]. Subsequently, 10 µL of the 0.22 µm-filtered wastewater samples were individually mixed with equal volumes of double-strength BHI broth (supplemented with 2 mM CaCl 2 ) and 100 µL of mid-log cultures of either S. Typhimurium strain 85 or S. Enteritidis strain FORC_052. The enriched tubes were incubated for 48 h at 37 • C with continuous agitation at 100 rpm, and then spun down at 10,000× g for 10 min at 4 • C. The supernatants were collected and filtered through 0.22 µm millipore syringe filters.
The presence of bacteriophages was evaluated by spotting 10 µL of the enriched lysates onto lawns of either S. Typhimurium strain 85 or S. Enteritidis strain FORC_052, as previously described [43]. Culture dishes, spotted with phage, were examined for the presence of clear areas following overnight incubation at 37 • C. The lytic area was then cut from the top surface and immersed into 500 µL salt-magnesium (SM) buffer (0.1 M NaCl, 0.05 M Tris-HCl, and 0.01 M MgSO 4 ; pH 7.5). Phage particles were allowed to diffuse out into the SM buffer through overnight incubation at room temperature with continuous shaking.

Phages Purification and Propagation
The purification of the isolated phages was conducted by the double agar overlay (DAO) method [44]. Single plaques showing distinct plaques morphotypes were collected from the top agar surface using sterile micropipette tips, resuspended in SM buffer, and maintained at 4 • C for 2 h. Again, the suspensions were plated using the DAO method and this procedure was repeated until morphologically identical plaques were obtained.
The full-plate lysis method was employed to propagate the purified phages [45]. The purified phages were tenfold serially diluted in SM buffer and plated using the double agar overlay method and incubated for 24-48 h at 37 • C. Plates that showed full lysis were selected and 4 mL of SM buffer was poured over the lysed area and incubated overnight at 4 • C with continuous shaking at 100 rpm. Phage suspension in the supernatant was collected by aspiration, vortexed, and centrifuged at 10,000× g to remove any host debris. The propagated phages were filtered through 0.22 µm and phages concentrations (PFU/mL) were evaluated using the agar overlay method.

Determination of Phages Host Range
The spectrum of killing activity of STP11 and STP13 were conducted against selected bacterial isolates by the spot assay [48]. Subsequently, 100 µL of an exponential phase culture (~10 8 CFU mL −1 ) was mixed with 5 mL of molten BHI soft agar (0.6% agar). The preparation was then poured onto nutrient agar plates. Once dried for 5 min, 5 µL phage lysate was placed onto the top agar layer and kept at room temperature for adsorption for 20 min. The plates were then incubated overnight at 37 • C. Consequently, the plates were inspected for the presence of a growth-free area, and its presence was reflected as positive for the test, which would be further confirmed using the DAO method.

One-Step Growth Curve
The intracellular lytic activities of the isolated phages in a one-round replication cycle, following the procedure displayed by Bloch et al. [49], with minor modifications. The host bacterium was grown in a BHI medium at 37 • C with shaking until OD 600 = 0.2. Afterward, 10 mL of the culture sample was spun down (4000× g) for 10 min at 4 • C. Following that, the pellet was resuspended in fresh LB medium. A fixed number of each bacterial host cell (5 log 10 CFU/mL) in the mid-log-phase were inoculated with their corresponding phages at an MOI of 10. Adsorption was allowed for 5 min at 37 • C while shaking at 140 rpm, non-adsorbed virions were eliminated by rinsing 3 times with 1000 µL of BHI medium containing 3 mM sodium azide (4 • C, 4000× g, 10 min). Next, the pellet suspension was mixed with 25 mL of BHI medium (time 0) and incubated in a shaker incubator at 37 • C. Over the course of 60 min, approximately 100 µL aliquots were collected at intervals of 10 min. The DAO method was performed to obtain the phage titer of these aliquots after diluting them with phage buffer. Finally, the period of latency and the magnitude of virions released from one cycle (burst size) were determined. This experiment was repeated three times.

Thermal and pH Stability Assay
The thermal resistance of all bacteriophages was determined by heating the isolated phages at 40 • C-90 • C in a temperature-controlled water bath, and stability at 4 • C was performed in a standard refrigerator. Equal volumes of the purified phage lysate (10 8 PFU/mL) and PBS (pH = 6.5) were incubated for 2 h. The influence of pH on the bacteriophages was assayed in nutrient broth at a pH range of between 2.0 and 14.0. The experiments were conducted at 4 • C for 2 h. The thermal and pH stabilities were determined by measuring the residual phages (PFU/mL) using the DAO technique [45].

Killing Assay
The lytic efficacy of the isolated phages were determined, as previously described [43]. Subsequently, overnight cultures of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052 were diluted to 10 5 CFU/mL; then, the diluted cultures were challenged individually with their corresponding phages at MOIs of 100 and 10,000. The mix was then incubated for 24 h at 37 • C. The aliquots were harvested at 0, 2, 4, 6 and 24 h post-infection (p.i.) and were serially diluted to count the surviving Salmonella cells.

Organic and Detergent Solvents
The effects of organic solvent and disinfectants on the stability of phages STP11 and STP13 were performed according to Jurczak-Kurek et al.'s protocol, with slight modifications [50]. For each solvent, 1000 µL (6 × 10 7 PFU/mL) of phage particles were mixed with equal volumes of Sodium hypochlorite (NaOCl, 6% v/v) and organic solvents (70% ethanol) (Sigma Aldrich, St. Louis, MO, USA), separately, and incubated for 1 h, at 37 • C with gentle shaking. The untreated controls were prepared by mixing equal volumes of phage lysates with PBS (pH 7.4) under the same conditions. The mixtures were centrifuged at 9000 rpm for 12 min, and the phage titer was determined by the DAO method.

Genomic Characterization of the Isolated Phages
The genomic DNAs of phages STP11 and STP13 were extracted, purified, and quantified using the Phage DNA Isolation Kit (Biotek Corp, Norgen, ON, Canada), as per the manufacturer's protocol. The DNA quantities were estimated using a NanoDrop ND-1000 UV-Vis spectrophotometer. The isolated DNA were stored at −20 • C for further analysis.
The purified phages' DNA were sent to the microbial genome sequencing center (Pittsburgh, PA, USA) for sequencing. The phage genome was sequenced using a TruSeq protocol on an Illumina HiSeq platform, with 100 bp pair-end read sizes. FastQC was used to check the quality of the raw reads and the FASTQ Quality trimmer (minimum Q30 score) was used for trimming available on the public Galaxy server (https://usegalaxy.org/. Accessed on 15 August 2022). The trimmed reads were de novo assembled to a single contig with 120-fold coverage using Geneious 9.0.5 [51].
The genome map was constructed using the BLAST Ring Image Generator (BRIG) platform and the CGView online tool was used to estimate the GC content and GC skew of the genome [52]. The PHIRE platform was used to generate the promoters which are specific for the DNA sequence of the isolated phages [53].

Phylogenetic Analysis
The nucleotide sequence alignment and phylogenetic analysis were carried out using ClustalW and the Neighbor-joining method, employing MAFFT v.7 software [64]. The phylogenetic tree was visualized in iTOL [65].

Genome Comparison in a Two-Dimensional Plot
The Vector Builder's Sequence Dot Plot tool (https://en.vectorbuilder.com/tool/ sequence-dot-plot.html) was utilized to investigate close similarity genomic regions between the isolated phages sequence in comparison with the reference sequence selected from the national database (NCBI), which showed a high percent identity to the isolated phages. The two sequences were compared in a two-dimensional plot and organized on the left Y and top X axes of a two-dimensional matrix; the green and red dots represent the coordinates at which both sequences match.

Statistical Analysis
Statistical analysis was conducted using one-way analysis of variance (ANOVA) with the aid of GraphPad Prism software version 6 for windows (GraphPad Software Inc. San Diego, CA, USA). Statistical significance was determined at p < 0.05.

Antimicrobial Sensitivity
The antibiotic resistance profiles of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052 were evaluated against a selection of antibiotics (n = 15) belonging to eight different classes ( Table 1). The data showed a resistance percentage of 46.6% and 66.6% for S. Typhimurium strain 85 or S. Enteritidis strain FORC_052, respectively, against the tested antibiotics. The tested bacteria were sensitive to the tested antibiotics belonging to the 3rd generation Cephalosporins and Carbapenems. The antibiogram data identified S. Typhimurium strain 85 and S. Enteritidis strain FORC_052 as multidrug-resistant (MDR) as they resisted many antibiotics belonging to different classes. Table 1. Antibiotic sensitivity profile of S. Enteritidis strain FORC_052 and S. Typhimurium strain 85 against a selection of fifteen antibiotics.

66.6%
Diameter of inhibition zones against the tested antibiotics were measured in millimeters (mm), bacterial isolates were reported as resistant (R, orange cells) or susceptible (S, green cells).

Bacteriophages Isolation and TEM Characterization
In this study, we isolated two Chi-like Salmonella phages, STP11 and SEP13, respectively, against the MDR S. Typhimurium strain 85 and S. Enteritidis strain FORC_052. The presence of phages in the collected raw wastewater samples was first screened by spot assay and then further confirmed by the double agar overlying method ( Figure 1A,B). A double agar overlying test was performed to determine the morphology of the plaques. STP11 and SEP13 phages produced clear, uniform-size plaques with diameters of 1.5 ± 0.5 mm and 0.5 ± 0.5 mm, respectively, on lawns of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052.
The TEM observation of phages STP11 and SEP13 showed similar morphotypes, including isometric capsids with long non-contractile tails, which are features of phages belonging to the Siphoviridiae family under order Caudovirales ( Figure 1C,D). Phage STP11 displayed an icosahedral head with a diameter of 65.6 ± 1.4 nm and a flexible, non-contractile tail (227 ± 1.5 nm length, 12 ± 1.5 nm width). Similarly, phage SEP13 displayed the same head and tail morphology with a capsid diameter of (64 ± 1.2) nm and a tail length of 226 ± 1.2 nm, and 11 ± 1.5 nm in width, respectively. mm and 0.5 ± 0.5 mm, respectively, on lawns of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052.
The TEM observation of phages STP11 and SEP13 showed similar morphotypes, including isometric capsids with long non-contractile tails, which are features of phages belonging to the Siphoviridiae family under order Caudovirales ( Figure 1C,D). Phage STP11 displayed an icosahedral head with a diameter of 65.6 ± 1.4 nm and a flexible, non-contractile tail (227 ± 1.5 nm length, 12 ± 1.5 nm width). Similarly, phage SEP13 displayed the same head and tail morphology with a capsid diameter of (64 ± 1.2) nm and a tail length of 226 ± 1.2 nm, and 11 ± 1.5 nm in width, respectively.

Sensitivity of the Isolated Phages to Physical and Chemical Agents
The thermal stabilities of the isolated phages were assayed at neutral pH (pH 7.0). The average titers of the two phages were found to be stable (7.8 log10 PFU/mL) upon exposure at 4 °C, 20 °C, 40 °C, or 60 °C for 2 h. The survival rate of STP11 declined to approximately 3.7 log10 PFU/mL (p < 0.05) at 80 °C. The titer of phage SEP13 decreased to 4.5 log10 PFU/mL at 70 °C. We observed that almost no phages survived (p < 0.05) at 90 °C and 80 °C for phages STP11 and SEP13, respectively (Figure 2A).
Concerning the pH stability, as shown in Figure 2B, phages STP11 and SEP13 retained a high titer (8 log10 PFU/mL) from pH 4 to pH 9 for 2 h. Phage STP11 showed a significant decrease (p < 0.05) in titer at pH 2 and 3, with phage titers of 2.5 and 1.85 log10 PFU/mL, respectively, while the titer of SEP13 decreased by 1.3 log10 PFU/mL at pH 3. The titer of both STP11 and SEP13 became zero at strong alkaline conditions specifically at pH

Sensitivity of the Isolated Phages to Physical and Chemical Agents
The thermal stabilities of the isolated phages were assayed at neutral pH (pH 7.0). The average titers of the two phages were found to be stable (7.8 log 10 PFU/mL) upon exposure at 4 • C, 20 • C, 40 • C, or 60 • C for 2 h. The survival rate of STP11 declined to approximately 3.7 log 10 PFU/mL (p < 0.05) at 80 • C. The titer of phage SEP13 decreased to 4.5 log 10 PFU/mL at 70 • C. We observed that almost no phages survived (p < 0.05) at 90 • C and 80 • C for phages STP11 and SEP13, respectively (Figure 2A).
Concerning the pH stability, as shown in Figure 2B, phages STP11 and SEP13 retained a high titer (8 log 10 PFU/mL) from pH 4 to pH 9 for 2 h. Phage STP11 showed a significant decrease (p < 0.05) in titer at pH 2 and 3, with phage titers of 2.5 and 1.85 log 10 PFU/mL, respectively, while the titer of SEP13 decreased by 1.3 log 10 PFU/mL at pH 3. The titer of both STP11 and SEP13 became zero at strong alkaline conditions specifically at pH 13 and pH 14, respectively (p < 0.05). Similarly, there are no viable virions encountered at pH 1 for both phages.
The stability of the isolated phages against chemical agents was determined by subjecting it to Sodium 6% hypochlorite (NaOCl) and 70% ethanol. The results obtained from this experiment indicated that the same results were attained for both phages. According to the results, more than half of the titer of both STP11 and SEP13 was maintained at Sodium hypochlorite treatment. However, their titer was equally reduced to approximately 5.5 and 7.3 log 10 PFU/mL in the presence of 70% ethanol and NaOCl at 60 min post-treatment, respectively, compared to the control (p < 0.05) ( Figure 2C).
13 and pH 14, respectively (p < 0.05). Similarly, there are no viable virions encountered at pH 1 for both phages.
The stability of the isolated phages against chemical agents was determined by subjecting it to Sodium 6% hypochlorite (NaOCl) and 70% ethanol. The results obtained from this experiment indicated that the same results were attained for both phages. According to the results, more than half of the titer of both STP11 and SEP13 was maintained at Sodium hypochlorite treatment. However, their titer was equally reduced to approximately 5.5 and 7.3 log10 PFU/mL in the presence of 70% ethanol and NaOCl at 60 min post-treatment, respectively, compared to the control (p < 0.05) ( Figure 2C).

One-Step Growth Curve
One-step growth kinetics were performed to determine the latent periods and the burst sizes of the isolated phages (

One-Step Growth Curve
One-step growth kinetics were performed to determine the latent periods and the burst sizes of the isolated phages (

Bacterial Challenge Test
The efficacy of phages STP11 and SEP13 to control the growth of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052, respectively, was determined ( Figure 4). In comparison to the unchallenged bacterial counts, the results showed that both phages evidently restrained the growth of their corresponding hosts, 2 h post-infection (below the detection limit, <1 log10 CFU/mL), when the cells were challenged with an MOI of 10,000. However, at MOI of 100, bacterial growths were inhibited for up to 6 h post-infection; then, the bacterial counts were increased gradually after 24 h p.i. This rise in survival is likely due to the rise of mutant variants early on in the growth curve, as at high MOIs all the cells become infected and the resistant ones are unaffected and quickly amplify in the nutrient-rich medium.

Bacterial Challenge Test
The efficacy of phages STP11 and SEP13 to control the growth of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052, respectively, was determined ( Figure 4). In comparison to the unchallenged bacterial counts, the results showed that both phages evidently restrained the growth of their corresponding hosts, 2 h post-infection (below the detection limit, <1 log 10 CFU/mL), when the cells were challenged with an MOI of 10,000. However, at MOI of 100, bacterial growths were inhibited for up to 6 h post-infection; then, the bacterial counts were increased gradually after 24 h p.i. This rise in survival is likely due to the rise of mutant variants early on in the growth curve, as at high MOIs all the cells become infected and the resistant ones are unaffected and quickly amplify in the nutrient-rich medium. Figure 3. One-step growth kinetics of phages STP11 and SEP13 on their corresponding hosts. Results are displayed as means of three replicates ± SD and presented as log10 (PFU/mL).

Bacterial Challenge Test
The efficacy of phages STP11 and SEP13 to control the growth of S. Typhimurium strain 85 and S. Enteritidis strain FORC_052, respectively, was determined ( Figure 4). In comparison to the unchallenged bacterial counts, the results showed that both phages evidently restrained the growth of their corresponding hosts, 2 h post-infection (below the detection limit, <1 log10 CFU/mL), when the cells were challenged with an MOI of 10,000. However, at MOI of 100, bacterial growths were inhibited for up to 6 h post-infection; then, the bacterial counts were increased gradually after 24 h p.i. This rise in survival is likely due to the rise of mutant variants early on in the growth curve, as at high MOIs all the cells become infected and the resistant ones are unaffected and quickly amplify in the nutrient-rich medium.

Host Range
The host range of STP11 and SEP13 was assessed by a spot test and confirmed by plaque assay. The results revealed that out of the 14 strains tested, phage STP11 was lytic against 42.8% (n = 6), while SEP13 showed lytic activity against 35.7% (n = 5) of the tested bacterial spp. The lytic activities of both phages were limited to the targeted bacterial spp. (Table 2).

Host Range
The host range of STP11 and SEP13 was assessed by a spot test and confirmed by plaque assay. The results revealed that out of the 14 strains tested, phage STP11 was lytic against 42.8% (n = 6), while SEP13 showed lytic activity against 35.7% (n = 5) of the tested bacterial spp. The lytic activities of both phages were limited to the targeted bacterial spp. (Table 2).

Genomic Features
Phages STP11 and SEP13 have linear dsDNA genomes consisting of 58,890 bp and 58,893 bp nucleotide sequences with a G + C content of 57% and 56.5%, respectively ( Figure 5). Whole genome sequences of both phages STP11 and SEP13 were deposited in the GenBank database under accession numbers OP535471 and OP535472, respectively. Bioinformatics analysis revealed that the genome of phage STP11 ( Figure 5A) and SEP13 ( Figure 5B) contained 70 and 71 ORFs, respectively. No gene encoding tRNA was detected in their genome. Of the 70 putative ORFs of phage STP11, 27 (38.6%) were assigned to functional genes and 43 (61.4%) were annotated as hypothetical proteins (Table 3). Similarly, 29 (40.8%) of the 71 putative ORFs of phage SEP13 were annotated as functional genes, whereas the remaining 42 (59.2%) were assigned as nonfunctional proteins. Out of the 70 putative ORFs of phage STP11, 26 (37.1%) ORFs were on the negative strand, while the other 44 (62.9%) ORFs were on the positive strand. In the genome of phage SEP13, 24 (33.8%) ORFs were situated on the positive strand, while the remaining 47 (66.2%) ORFs were found on the negative strand. In the case of the STP11 genome, most of the annotated ORFs began ATG as a starting codon, with the exception of ORFs (3,29,52), which began with GTG, and ORF58 which began with CTG. Detailed information regarding the annotation is provided in Supplementary Table S1. The majority of SEP13's ORFs began with the ATG codon, with the exception of ORF68, ORF43, and ORF1, which began with GTG, CTG, and GCG, respectively (Supplementary Table S2).    The predicted functional proteins were categorized into five modules: packaging, DNA metabolism (DNA replication and encapsulation), host lysis, head, tail morphogenesis, and other proteins. The head-tail associated proteins of phage STP11 were represented by capsid scaffolding protein, major capsid protein, prohead protease ClpP, decorator protein D, putative tape measure protein, phage tail protein, and tail tape measure protein, which were encoded by ORF31, ORF10/ORF32, ORF8, ORF9, ORF19, ORF23, and ORF20/ORF44, respectively. Similar proteins were encoded by ORF69, ORF18, ORF20, ORF19, ORF11, ORF6, and ORF57/ORF11, respectively. In these functional protein categories, the putative tail fiber protein of SEP13 was encoded by ORF5. The genome of both phages STP11 and STP13 encoded three DNA replication proteins: XRE family transcriptional regulator, replication protein DnaD, and putative N-6-adenine-methyltransferase; however, in contrast to phage STP13, the genome of phage STP11 encoded three DNA replication proteins: helicase family protein, putative DNA polymerase, and Deoxyribosyl transferase, represented by ORF25, ORF27 and ORF67, respectively. Putative lambda family portal protein B and terminase large subunit were encoded by ORF7 and ORF24 for phage STP11 and ORF21 and ORF23 for phage STP13, respectively. In contrast to STP11, the genome of phage SEP13 was encoded for two cell lysis proteins: endolysin and putative endolysin 2 proteins, represented by ORF52 and ORF71, respectively. In addition to the above-mentioned functional proteins, other accessory proteins were encoded by the genome of both phages (Table 3).
The Vector Builder's Sequence Dot Plot tool was used to determine the degree of the close similarity between phage STP11 (Supplementary Figure S1A) and SEP13 (Supplementary Figure S1B) in comparison with the reference sequence of Salmonella phages ST-374 (NC_052998.1) and ER24 (NC_052999.1), selected from the national database, which showed high query coverage, accession length, and high sequence similarity (95% and 97.15%, respectively). The two-dimensional matrix confirmed that the reference and the isolated sequence showed a high sequence match (100%) in the majority of the genomic regions, as indicated by green and red dots for both reverse and forward sequences, respectively (Supplementary Figure S1).
The phylogenetic analysis relied on the major capsid protein and indicated that the isolated phages (STP11 and SEP13) showed high sequence similarity to each other and to other Chi-like salmonella phages, whilst showing evolutionary distant from the nonsalmonella Chi-like phages ((Providenca phage PSTCR9 (QPB12562.1)), Providenica phage Redjac (YP 006906019.1), Klebsiella phage Seifer (YP 009841554.1), Aeromonas phage vB AhyS-A18P4 (YP 009998227.1)) ( Figure 6).  Figure 6. Phylogenetic tree was made using the whole genome sequence (A) and amino acid sequence of the major capsid protein (B) of phage STP11 and SEP13 and phages sharing homology sequence identity retrieved from GenBank (NCBI). The sequences were aligned using ClustalW, and the tree was built using MEGA 7 software. The evolutionary history of 33 major capsid protein amino acid sequences and 37 core genes of the whole genome sequence were aligned and inferred using the Neighbor-Joining method and 1000 bootstrap replicates. The red dot highlights the isolated phages. The scale bar represents 20% nucleotide substitution and 10% amino acid substitution percentage for the whole genomic and capsid protein map, respectively.

Discussion
Salmonella phages are used in many lab-oriented applications, including the creation of strains through the process called transduction [66,67], and typing them for Figure 6. Phylogenetic tree was made using the whole genome sequence (A) and amino acid sequence of the major capsid protein (B) of phage STP11 and SEP13 and phages sharing homology sequence identity retrieved from GenBank (NCBI). The sequences were aligned using ClustalW, and the tree was built using MEGA 7 software. The evolutionary history of 33 major capsid protein amino acid sequences and 37 core genes of the whole genome sequence were aligned and inferred using the Neighbor-Joining method and 1000 bootstrap replicates. The red dot highlights the isolated phages. The scale bar represents 20% nucleotide substitution and 10% amino acid substitution percentage for the whole genomic and capsid protein map, respectively.

Discussion
Salmonella phages are used in many lab-oriented applications, including the creation of strains through the process called transduction [66,67], and typing them for epidemiological studies [68]. The specificity of some Salmonella phages and their peptides has also been used to produce strain or species-specific bio-probes for the quick detection of Salmonella on different food matrices [69,70] and as antibiotic alternatives to eradicate different salmonella strains on foods including chicken carcass [71,72]. In this study, we isolated novel Chilike Salmonella phages from samples collected from the Jeddah wastewater treatment plant. Most Chi-like phages infect Salmonella enterica serovars, however, some of them are reported to be infectious for Providencia species [33] or Enterobacter species. Unfortunately, the presence of lysogenic genes in the genome of STP11 and SEP13 and related Salmonella Chi-like phages is the main drawback that prevents the use of these phages as therapeutic and/or biocontrol agents [73] Nevertheless, the recent advancement in phage genetic engineering allows scientists to generate strictly lytic phages using lysogenic phages [74] for diagnostic and clinical applications [75], including phage therapy in humans [76]. Phages STP11 and SEP13 belonged to the family Siphoviridae. Both STP11 and SEP13 phages showed comparable genome size and high sequence similarity with the Salmonella Chivirus. The genomic size of phage Chi is roughly 59 kb long with 75 ORFs and 56.5% GC content [31,32]. Related Chi-like phages with identical genome sizes, gene contents and orders to phage Chi include Salmonella phages FSL_SP-039, FSLSP030, FSLSP088, SPN19, FSL_SP-124 [31] Providencia stuartii phage RedJac [33] and Enterobacter cancerogenus phage Enc34 [34].
Successful phage therapy may depend on phage virulence, latent period, host range, burst size, obligatory lytic activities, and so on. Multiple bacterial infections are usually achieved by using broad host range phages. Some Salmonella bacteriophages have a wide host spectrum, but most show narrow host specificity that only infects its indicator host [54]. In this study, we found that, in comparison to phage SEP13, phage STP11 had relatively broad spectrums of antibacterial activity against the tested bacterium. With the exception of the indicator host, S. Typhimurium, STP11 showed the potential to infect Salmonella enterica subsp. arizonae, Salmonella enterica subsp. enterica serovar Dublin, S. Typhimurium (ATCC 14028), and Salmonella enterica subsp. enterica serovar Typhi.
According to the one-step growth cycle conducted in the present study, the isolated phages showed high burst sizes with short latent periods. According to the previous reports that the latent periods of STP11 and SEP13 were higher than the flagellotropic phage, iEPS5 (15 min) [37], but lower than the Chi-like viruses (STm101 and STm118) (>30 min) [55,77]. Phage STP11 and SEP13 had a higher burst size compared to STm101 (112 pfu/infected cell) and STm118 (48 pfu/infected cell) [77]. The use of phages with high lytic activity against large numbers of targeted bacterial populations is crucial for the large-scale biocontrol of host bacterium. This property is correlated with the large burst size. Having a large burst size for an antimicrobial agent is among the key characteristics of a good bacteriophage as burst size closely relates to phage propagation [56]. Large burst-size phages may have a selective advantage as an antibacterial agent as they can dramatically increase the initial dose several hundred-fold in a short period of time [73,78]. It is thus evident that a large burst size is a decisive advantage for their use as biocontrol agents against the tested strains.
Phage STP11 and SEP13 appeared to be stable under a broad range of temperatures (4-70 • C/80 • C) and pH values (3-12/13). These two phages did not show a significant loss of their titer for a 2 h incubation period between 4 • C to 60 • C. This finding is in agreement with the recent novel Salmonella Phage LPST153, which was isolated from a lake in China [79]. The two phages showed good stability at alkaline pH (pH 12), whereas reported titers of other phages were almost completely deactivated at pH 12 [80,81].
Phylogenomic analysis was conducted to investigate the relationship between our isolated phages and formerly reported Chi-like phages. In this regard, phages STP11 and SEP13 formed a monophyletic clade with each other and other Chi-like Salmonella phages, such as Salmonella phage vB SentM sal3 (MT499898.1), enterobacteria phage Chi (NC 021315.1), Salmonella phage 35 (NC 048632.1), and Salmonella phage ST-101 (NC 048648.1). The phylogenetic tree also indicated that the two candidate phages were phylogenetically distant from the non-Salmonella Chi-like phages. The constructed phylogenomic tree was not based on the whole genome sequence; rather, it was constructed using the 37 core genes. Hence, it may not accurately reflect their relationship. According to a report released by [61], core gene-based phylogenetic analysis represents the relationship between phages only in the high-gene flux mode. Phage-mediated horizontal gene transfer may result in genomic variation, which can obscure evolutionary relationships among phages [82,83]. Moreover, phages lack a conserved marker, universal genes, which makes it difficult to study the origin and evolutionary relationship of phages [84].

Conclusions
There have been several Chi-like Salmonella phages isolated so far. However, detailed molecular, as well as proteomic, studies are lacking. In this study, we have isolated and characterized two Chi-like Salmonella phages that were isolated using two different hosts. Based on the whole genomic sequence analysis and physicochemical parameters, the two phages shared some common characteristics which are the features of Chi-like phages. Taking into consideration the molecular analysis, the identification of specific proteins which determine the infection cycle will be crucial to broadening our understanding of these unusual phages and their interaction with the host cells. Moreover, further studies are needed to convert these phages to obligatory lytic phages by removing the lysogenic genes for better biocontrol uses.